The regulation of transcription is a key step in many biological processes in eukaryotic cells, including control of cell growth and division, differentiation of tissues and development of organs, and response to extracellular signals. Transcriptional regulation is also critical to the expression of the genetic programs of many eukaryotic viruses, including the well-known examples of SV40, adenoviruses, and herpesviruses. In our effort to understand how the initiation of transcription is regulated, we study the VP16 protein of herpes simplex virus type 1 (HSV-1), which has been widely adopted as a paradigm for eukaryotic transcriptional activation. The long-term objective for the project supported by this Research Career Development Award is an understanding of the molecular details by which VPI6 activates transcription of the viral IE genes by the host RNA polymerase II. In this project period, we will pursue that objective with five specific aims. 1. We will identify amino adds in the VP16 activation domain that are critical for its transcriptional function. We have already identified a number of amino acids critical to the function of a subdomain of VP16 (residues 413-456). We have recently shown that residues 450-490 constitute an independent activation domain that utilizes a much different pattern of amino acids. We will employ site- directed mutagenesis, alanine-scanning mutagenesis, and random mutagenesis with genetic selection to identify and further characterize the critical amino acids for the two distinct subdomains of the VP16 transcriptional activator. 2. We will define the interactions between the VP16 activation domain and its various putative target proteins. Biochemical and genetic methods have been used to identify putative target proteins for transcriptional activators, including the basal transcription factors TFIID (both the TATA-binding protein, TBP, and an associated TAF protein), THIIB, and TFIIH; adaptor proteins, such as ADA2 and ADA3; and even proteins known for their role in replication (e.g., RPA). We will characterize a number of these interactions by employing our collection of VP16 mutations in biochemical, biophysical, and genetic assays. 3. We will explore the secondary and tertiary structure of the VP16 activation domain. Previous reports and unpublished results show that the activation domain of VPl6 is largely unstructured in solution. Preliminary results using fluorescence and NMR spectroscopy suggest that the domain may become more ordered in the presence of putative target proteins. The structure of VPI6 will be pursued using these and related methods, in collaboration with investigators having distinguished reputations in these fields. 4. We will characterize the transcriptional activation domains of VP16 homologs from related herpesviruses. Viruses related to HSV-1 are significant pathogens of humans and agriculturally important animals. We have used a novel protein sequence analysis method to identify putative transcriptional activation domains in VP16 homologs from these viruses. Preliminary observations on the ORF10 gene product of varicella-zoster virus, in collaboration with Jeffrey Cohen and colleagues, have validated this approach. We will apply this method to identify and characterize the activation domains of additional homologs. 5. We will evaluate the role of transcriptional activation by VP16 during the lytic infection process. Transcriptional activation by VP16 has been primarily investigated using methods in which the VP16 gene and protein are explanted from their biological sites in the viral genome and virion, respectively. We have now constructed recombinant viruses bearing deletion mutations of part or all of the VP16 activation domain. We will examine the effects of these mutations on the growth of the virus in culture, and upon the transcriptional activation of viral IE genes during lytic infection in culture.